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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR STRATEGIES FOR DETECTION AND IDENTIFICATION OF NEMATODES INFECTING FORAGE LEGUMES AND DEVELOPMENT OF HOST RESISTANCE
2008 Annual Report


1a.Objectives (from AD-416)
Objective 1: Discover, refine, and implement improved molecular approaches to modernize classification and aid the identification and control of nematodes in alfalfa cropping systems. Objective 2: Identify and develop novel sources of resistance to nematodes.


1b.Approach (from AD-416)
1. Molecular markers, including ribosomal, mitochondrial, Hsp90, and other nuclear genes will be used to develop new diagnostic assays, including RFLPs, and conventional or real-time PCR assays for Meloidogyne spp., Pratylenchus spp., and Ditylenchus spp. 2. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 3. Bioinformatic analysis of EST sequences of selected species will be used to uncover genes that meet selective criteria for use as diagnostic markers and for phylogenetic comparisons. Selected gene sequences will be used to design oligonucleotide primers for amplification,in polymerase chain reactions, of specific gene sequences from populations of phytopathogenic nematodes to evaluate them for genus level, species-wide, and species-specific identification. 4. Novel gene targets from Meloidogyne spp. and Pratylenchus spp. will be advanced through a pipeline of cloning, mRNA expression profiling, and functional characterization, leading to identification of specific genes with the best potential for further development into novel control methods. 5. Based on the information obtained above, selected genes will be targeted for silencing by gene-specific dsRNA; nematodes treated with dsRNA will be monitored for the ability to move, infect host plants, feed and reproduce. Effects on gene expression will be monitored by mRNA extraction and PCR using gene-specific primers directed at the gene to be silenced. Genes that show the most promising phenotypes in the soaking experiments will be transformed into M. truncatula hairy roots using Agrobacterium-mediated transformation methods. Transgenic plants containing target gene RNAi will be infected with root-knot or lesion nematodes and assessed for decreases in nematode infection and reproduction; knock out of target genes will be verified through RT-PCR and in situ detection methods.


3.Progress Report
Molecular Diagnostics and Phylogeny of Nematodes: Extensive analysis of multiple DNA markers (rDNA and heat shock protein 90, Hsp90) were used to identify and describe an unusual non-galling root-knot nematode (Meloidogyne arenaria) isolated from a diseased palm-like ornamental tree (traveler's tree) from Florida, and to characterize three populations of stunt nematode (parasitic on grasses, alfalfa, and other crops) for integration with phylogenies based on anatomical features. A database of Hsp90 DNA sequences was expanded to include several additional populations of potato cyst and tobacco cyst nematodes to facilitate discovery of urgently needed new diagnostic markers. The effect of alternative new storage methods on PCR amplification from single nematode genomic DNA were examined for the purpose of improving the long-term stability of archived specimens. This research addresses National Program 215 Rangeland, Pastures and Forages, Component III (Sustainable Harvested Forage Systems for Livestock, Bioenergy, and Bioproducts) Problem Area H: Need for improved forage legumes that can be grown, harvested, and used in an economically and environmentally sound manner. These project objectives are also relevant to National Program 303, Plant Diseases, Component I.


4.Accomplishments
1. Description of pale potato cyst nematode in the U.S.

Cyst nematodes are an important group damaging the roots of many kinds of plants, including crops grown in rotation with alfalfa. In the present study, ARS scientists in Beltsville, MD described the anatomical and molecular characteristics of a pale potato cyst nematode from soil associated with potato fields in eastern Idaho, providing the first detailed description of this nematode in the U.S. This research is significant because it describes a new diagnostic test that can distinguish pale potato cyst nematode from look-alike cyst nematodes. Because the pale cyst nematode is regulated as a quarantine pest by many countries and causes economic damage to potato worldwide, scientists, regulators, and extension agencies will use this research to better identify and prevent the spread of this nematode. This research addresses National Program 215 Rangeland, Pastures and Forages, Component III (Sustainable Harvested Forage Systems for Livestock, Bioenergy, and Bioproducts) Problem Area H: Need for improved forage legumes that can be grown, harvested, and used in an economically and environmentally sound manner. These project objectives are also relevant to National Program 303, Plant Diseases, Component I: Detection, Identification and Characterization of Plant Pathogens and Component IV: Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings2
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2
Number of Other Technology Transfer2

Review Publications
Skantar, A.M., Handoo, Z.A., Carta, L.K., Chitwood, D.J. 2007. Morphological and molecular identification of Globodera pallida associated with potatoes in Idaho. Journal of Nematology. 39(2):133-144.

Last Modified: 12/19/2014
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